The purpose of these studies is to define the various phases of respiratory tract carcinogenesis at the cellular and molecular level using in vitro systems. We have previously shown that the malignant transformation of rat tracheal epithelium could be studied in its entirety under well-controlled culture conditions. Subsequently we showed that we could enhance transformation using a well-known tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate thus demonstrating multistage carcinogenesis. This in vitro system has now been refined from organ-culture-exposed epithelium to monolayer-exposed epithelium thus allowing more quantitation and precision as to cell numbers at risk and cytotoxicity determinations. Early following carcinogen exposure we have found that there is a profound change in morphology and growth kinetics of these cells when compared to control. The elevation of growth and the percentage of cultures capable of continuous subculture was dose dependent. We also found that substrate requirements and nutritional requirements change markedly. These nutritional and hormonal requirements continue to change from the premalignant to malignant stages. We are currently utilizing these markers to investigate mechanisms of carcinogen dose response and tumor promotion in cultured airway epithelium.